Methods have been developed which will be used to examine specific reduced folate pools and polyglutamate conjugates thereof in mouse tissues and in cultural mouse hepatoma cells. The method is based upon incorporation of endogenous methylenetetrahydrofolate polyglutamate into a stable ternary complex with thymidylate synthetase and tritiated fluorodeoxyuridylate. Quantitation of the folate is measured by gel chromatography and polyglutamate chain length is measured by electrophoretic separation based on charge associated with complexed folylpolyglutamate. The method will specifically be used to develop an assay for folylpolyglutamate synthetase and polyglutamate hydrolase activities, examine degradation of endogenous folylpolyglutamate in cell-free tissue extracts, examine changes in the tetrahydrofolate and methylenetetrahydrofolate polyglutamates during the cell cycle of the cultured mouse hepatoma cells synchronized by leucine deprivation, and initiate experiments to observe changes in these and other specific reduced folate pools in mouse hepatoma cells after exposed to methotrexate.